The present invention relates in general to DNA molecules encoding a new polypeptide of the 11-12 transmembrane domain transporter family having a Mg2+- or Zn2+-proton exchange activity, expression vectors comprising them, plant cells transformed thereby and transgenic plants expressing same.
In all living organisms, cellular functions require a fine homeostasis of various ions and nutrients, including Mg2+ and Zn2+. Mg2+ is required for the function of manv enzymes (e.g., phosphatases, ATPases. RNA polymerases). Zn2+ plays both a functional (catalytic) and structural role in several enzyme reactions, and is involved in the regulation of gene expression by zinc-finger proteins. Both Mg2+ and Zn2+ are essential for the structural integrity of ribosomes. In plants, Mg2+ is also an essential component of chlorophyll, and regulates the activity of key chloroplastic enzymes.
Multicellular organisms have to balance not only their Mg2+ and Zn2+ intake and intracellular compartmentalization. but also the distribution of these ions to various organs. The movement of ions through membrane barriers is mediated by specialized proteinsxe2x80x94channels, transporters or ATPases. Thus far, genes encoding Mg2+ transporters have been cloned only from bacteria and yeast. The bacterial MgtA and MgtB Mg2+ transport proteins are P-type ATPases (Hmiel et al., 1989). Mg2+ is also transported by the bacterial CorA and mgtE proteins (Smith et al., 1993; Smith et al., 1995), but the molecular mechanism of Mg2+ mobilization by these proteins is not known. Among the Zn2+ transport proteins whose genes have been cloned, the bacterial ZntA (Rensing et al., 1997) is a P-type ATPase. Zn2+ is also transported by the yeast ZRT 1,2 (Zhao and Eide, 1996a; Zhao and Eide, 1996b), transporters, but the molecular mechanism of Zn2+ transport by these proteins is also unknown. A mammalian protein designated DCT1 (Gunshin el al., 1997), which belongs to the Nramp family of macrophage proteins, was suggested to be a symporter of protons with various divalent metal cations, including Fe2+ and Zn2+, but it was not able to symport Mg2+ ions.
Little is known about transport proteins that control. Mg2+ and Zn2+ homeostasis in plants. Ions absorbed into the cytosol of root cells diffuse towards the vascular cylinder through plasmodesmata and reach the xylem parenchyma cell layer, which border the xylem vessels. The xylem parenchyrna cells were suggested to play a key role in ion secretion into the xylem (xylem loading), and in the release of ions from the xylem (unloading). These processes require transport through the plasma membrane of the xylem parenchyma cells, but the proteins mediating xylem loading and unloading of Mg2+ and Zn2+ are not known. Unloaded Mg2+ and Zn2+ subsequently enter the surrounding cells through unknown transport proteins. The molecular mechanisms of phloem loading and unloading with Mg2+ and Zn2+ have also not been elucidated. Intracellularly, the vacuole is considered the main organelle mediating Mg2+ homeostasis in the cytosol and the chloroplast. Vacuolar Mg2+ is also important for the cation-anion balance and turgor regulation of cells. The activity of a Mg2+/H+ antiporter was identified in lutoid (vacuolar) vesicles of Hevea brasiliensis (Amalou et al., 1992; Amalou et al., 1994) and in vacuolar membranes from roots of Zea mays L. (Pfeiffer and Hager, 1993), but cloning of the corresponding genes has not been reported. The Hevea brasiliensis transporter was indicated to be electroneutral, and to be capable of transporting also Zn2+ cations. In Zn2+ tolerant species, tolerance is achieved mainly through sequestering Zn2+ in the vacuoles, but the transport mechanism is not known.
The progressive salinization of irrigated land threatens the future of agriculture in the most productive areas of our planet. Increasingly, intensive irrigation practices are resulting in secondary salinization of agricultural soils. Even water of good quality may contain 100-1000 g salt/m3. With an annual application of 10,000 m3/ha, between 1 and 10 t of salt are added to the soil. As a result of transpiration and evaporation of water, soluble salts further accumulate in the soil. Since crop productivity of irrigated land in many areas is much higher than of non-irrigated land, the coincidence of irrigation and salinization threatens current agricultural productivity. It has been estimated that 10xc3x97106 ha per annum of irrigated land are abandoned due to salinization and alkalization. For example, large areas of the Indian subcontinent have been rendered unproductive by salt accumulation and poor water management; in Pakistan, about 10 million of 15 million hectares of canal-irrigated land are becoming saline. Worldwide, about 33% of the irrigated land is affected by salinity, and presumably more land is going out of irrigation due to salinity than there is new land coming into irrigation.
Salinity problems occur also in non-irrigated croplands and rangelands either as a result of evaporation and transpiration of saline underground water or due to salt input from rainfall. The saline areas of the world consist of salt marshes of the temperate zones, mangrove swamps of the subtropics, and their interior salt marshes adjacent to salt lakes. Saline soils are abundant in semiarid and arid regions, where the amount of rainfall is insufficient for substantial leaching.
Soluble salts accumulating in the soil must be removed periodically by leaching and drainage. But even when proper technology is applied to the soils, they contain salt concentrations which often impair the growth of crop plants of low salt tolerance. Most crop species and cultured woody species either have a relatively low salt tolerance, or their growth is severely inhibited even at low substrate salinity. Salinity is the major nutritional constraint on the growth of wetland rice.
In saline soils, NaCl is usually the dominant salt. There are three major constraints for plant growth on saline substrate (Marschner, 1995, p. 662): (1) water deficit (xe2x80x98drought stressxe2x80x99) arising from the low (more negative) water potential of the rooting medium; (2) ion toxicity associated with the excessive uptake of mainly Clxe2x88x92 and Na+; (3) nutrient imbalance, caused by depression in uptake and/or shoot transport and impaired internal distribution of mineral nutrients, and calcium in particular.
In many fruit trees and herbaceous crop species, ion toxicity is characterized by growth inhibition and injury of foliage (marginal chlorosis and necrosis on mature leaves). These phenomena occur even at low levels of NaCl salination, under which water deficit is not a constraint. Many plant species such as citrus and leguminous suffer from Clxe2x88x92 toxicity. The species that suffer most from Na+ toxicity are graminaceous such as wheat, sorghum, and rice. Many crop species with relatively low salt tolerance are typical Na+ excluders, and are capable at low and moderate salinity levels of restricting the transport of Na+ into the leaves where it is highly toxic in salt sensitive species. The causes of salt toxicity in cells are inhibition of enzyme reactions and inadequate compartmentalization between cytoplasm and vacuole. There is also increasing support for the hypothesis of Oertli (1968) of salt accumulation in the leaf apoplasm as an important component of salt toxicity, leading to dehydration and turgor loss and death of leaf cells and tissues.
The mechanism of adaptation of plants to saline substrates is based on the principle that salt tolerance can be achieved by salt exclusion or salt inclusion. Differences in the capacity for Na+ and Clxe2x88x92 exclusion exist between cultivars of different species. For example, the higher salt tolerance of certain cultivars of wheat, barley and citrus is related to a more effective restriction of shoot transport of both Na+ and Clxe2x88x92. In grapevine, differences in salt tolerance are closely related to the capacity of rootstocks for Na+ and Clxe2x88x92 exclusion from the shoots. The capacity for Clxe2x88x92 exclusion seems to be the effect of a major dominant gene and appears to be independent of the ability of Na+ exclusion from the shoot. Mechanisms which restrict excessive Na+ and Clxe2x88x92 transport to the shoots of plants grown in saline substrates operate at root level (such as membrane properties, anatomical features) and along the pathway from roots to the shoot. It was shown that the stem tissue of certain species can reabsorbe Na+ from the xylem sap in periods of ample root supply. Retranslocation of Na+ from the shoots to the roots may also contribute to low Na+ contents in the shoots of certain species.
The movement of materials, including ions, in biological systems, particularly into and out of cells and across intracellular membrane barriers, is carried out by membrane proteins called transporters. In order to be integrated into the membrane, these transporters contain several hydrophobic domains, known as transmembrane domains or spans, which span on the membrane. Families of transporters are known with 11 or 12 transmembrane domains such as, for example, NCX1, a mammalian Na+/Ca2+ exchanger that plays a major role in extrusion of Ca2+ ions to the extracellular space following excitation (Nicoll et al., 1990).
According to the present invention, we have cloned and characterized an Arabidopsis transporter, herein designated MHX, of the amino acid sequence depicted in FIG. 1, a new member of the 11-12 transmembrane-domain transporter family that is localized in the vacuolar membrane and functions as an electrogenic exchanger of protons with Mg2+ and Zn2+ ions. The gene encoding MHX is the first gene encoding a Mg2+/H+ or Zn2+/H+ exchanger that has been cloned so far from any organism.
According to the present invention there is provided an isolated DNA molecule comprising a sequence encoding a polypeptide of the 11-12 transmembrane-domain transporter family having a Mg2+/H+ or Zn2+/H+ exchange activity.
The isolated DNA molecule of the invention may be a genomic, complementary or synthetic DNA. In one embodiment, the isolated DNA molecule is the complementary DNA (SEQ ID NOs:1 and 3) depicted in FIG. 2, or the genomic DNA (SEQ ID NO:4) depicted in FIG. 3, from Arabidopsis thaliana cv. C-24, coding for the 539-amino acid polypeptide MHX, a member of the 11-12 transmembrane-domain transporter family of the amino acid sequence (SEQ ID NOs: 2 and 3) depicted in FIG. 1. Hydropathy analyses using the Eisenberg, Schwarz, Komarony and Wall method revealed 11 putative transmembrane domains marked bold and underlined in FIG. 4, rendering MHX a member of the 11-12 transmembrane-domain transporter family.
Besides the shown Mg2+/H+ or Zn2+/H+ activity, MHX also has Fe2+/H+ exchange activity and may be expected to have an exchange activity for proton and other divalent cations such as cadmium, and it may also be involved in other processes in plants such as transport of monovalent cations such as sodium.
According to the present invention there is further provided a chimeric DNA molecule capable of expression in plants comprising: (a) a DNA molecule comprising a sequence encoding a polypeptide of the 11-12 transmembrane domain transporter family having a Mg2+/H+ or Zn2+/H+ exchange activity; and (b) DNA sequences capable of enabling the expression of said polypeptide in plant cells.
The DNA sequences of (b) capable of enabling the expression of said polypeptide in plant cells are, for example, a plant promoter and a plant polyadenylation and termination signal sequence at the 3xe2x80x2 non-translated region of the gene such as the nopaline synthase (nos) transcription terminator signal, and optionally a short DNA sequence at the 3xe2x80x2 end of the promoter for enhanced translation of the mRNA transcribed from the gene such as, for example, the omega (xcexa9) sequence derived from the coat protein gene of the tobacco mosaic virus (Gallie et al., 1987).
The promoter used according to the invention may be the natural MHX promoter or it is a DNA sequence not existing in nature linked to the MHX gene. The promoter may be a constitutive, organ-specific, tissue-specific, inducible or chimeric promoter. In one preferred embodiment, the promoter is the constitutive 35S promoter of cauliflower mosaic virus (CaMV35S).
According to the present invention there is further provided an expression vector comprising a chimeric DNA molecule of the invention. An example of such a chimeric DNA molecule is the construct depicted in FIG. 5 herein.
According to the present invention there is further provided a transformed plant cell expressing a polypeptide of the 11-12 transmembrane-domain transporter family having a Mg2+/H+ or Zn2+/H+ exchange activity.
According to the present invention there is further provided a transgenic plant whose cells express a DNA molecule comprising a sequence encoding a polypeptide of the 11-12 transmembrane-domain transporter family having a Mg2+/H+ or Zn2+/H+ exchange activity, particularly the MHX protein described herein, shown to have a divalent cation-proton exchange activity. Said transgenic plants are shown herein to have a lower content of sodium as compared with corresponding wild-type plants, and to have a higher dry matter weight upon growth in media with increased calcium levels as compared with corresponding wild-type plants. This makes them suitable for growth in calcareous soils, that are characterized by high calcium content that restrict plant growth.
The characteristics of the transgenic plants of the invention render them better adapted at growing under stress conditions. Thus, these transgenes will have an improved tolerance to stress conditions as compared with corresponding wild-type plants, said stress conditions comprising drought, temperature, mineral excess or deficiency, osmotic, pH, oxidant, chemical, pathogenic and, particularly, high salinity and high-calcium (saline and calcareous soils, respectively) stresses.